FIG 3.

Inhibition of autophagy pathway sensitizes Tax-expressing U251 cells to TRAIL-induced apoptosis. (A and B) U251 cells were infected with LV-EGFP or LV-Tax for 4 days, pretreated with 3-MA (5 mM or 10 mM), LY294002 (20 μM) (A), or 100 μM chloroquine (B) for 3 h, and then cultured for 12 h in the presence or absence of 50 ng/ml TRAIL. Cell viability was determined using MTS assays (mean ± SD; n = 3; *, P < 0.01 compared with TRAIL-treated Tax-expressing cells). (C) U251 cells were infected as for panels A and B, pretreated with 10 mM 3-MA for 3 h, and then treated with 50 ng/ml TRAIL for 6 h. The cell lysates were analyzed via Western blotting to determine the expression of c-FLIP, LC3, and Tax and the activation of caspase-8 and caspase-3. GAPDH was used as a protein loading control. Protein quantification was performed via densitometry, and the ratios of LC3-II to GAPDH are shown under the respective blots. (D) U251 cells were infected with LV-EGFP or LV-Tax for 12 h and then with LVs encoding Beclin-1 shRNA (shBECN1) or no shRNA (shCo) for another 12 h (MOI = 3). After 4 days, the cells were treated with 50 ng/ml TRAIL or PBS (control) for 12 h. Cell viability was determined via MTS assays (mean ± SD; n = 3; *, P < 0.01 compared with TRAIL-treated cells expressing Tax and shCo). The efficacies of BECN1 knockdown and LC3 expression are shown in the bottom panel. U251 cells were infected with LV-shCo or LV-shBECN1 for 4 days. The cell lysates were analyzed by Western blotting to determine the expression of BECN1 and LC3.